Amplification of nucleic acids and analysis of the resulting amplification products has revolutionized the basic and clinical sciences. Applications of these techniques include molecular cloning, nucleic acid sequencing, genotyping, detection and identification of single nucleotide polymorphisms (SNP) and other polymorphisms and mutations and the quantitation of gene expression,
Various techniques for nucleic acid amplification have been developed, such as strand displacement amplification, transcription-based amplification, and polymerase chain reaction (PCR).
Use of PCR in large scale research projects and in clinical applications entails amplification of many distinct target sequences with the concomitant generation of a great number of PCR amplicons. As the scale of such projects increases, it has become cost prohibitive and inefficient to undertake the necessary reactions singly. Thus, there is great interest in developing methods of performing multiple amplification reactions in parallel in the same reaction vessel using a common pool of template and reagents.
Such multiplex PCR methods, in which multiple pairs of target-specific primers are used to coamplify multiple targets, have met with only qualified success. Combining all the required primers in the same tube greatly increases the frequency of formation of primer-dimer and other spurious amplification products. As the number of primer pairs rises in multiplex PCR, the number of potential primer-dimer interactions (or spurious amplicons generated by two different primers) increases exponentially according to the number of primers used.
Even with careful attention paid to the design of the multiplex primer pairs to avoid obvious primer-dimer incompatibilities, conventional multiplex PCR is generally limited to about 10-20 simultaneous amplification reactions before undesired amplification products predominate.
Different approaches to ameliorate the problems associated with multiplex PCR have been developed, but none with unqualified success.
PCT publication WO 96/41012 discloses a method for multiplex PCR that entails two rounds of amplification and that uses primer pairs comprising template-specific sequences at their respective 3′ ends and universal, or common, primer sequences at their respective 5′ ends. The first round of amplification uses the specific primer sequences and the second amplification uses the universal primer sequences. The second round normalizes differential binding of the specific primers to different templates.
Another multiplex method uses a single specific primer for each target and a single common primer. N. E. Broude, et al., Multiplex allele-specific target amplification based on PCR suppression, Proc. Natl. Acad. Sci. USA 98:206-211 (2001). This method still suffers from the amplification of spurious products, however, and therefore remains limited in its application.
Thus, there remains a need in the art for methods of simultaneous multiplex amplification of large numbers of specific nucleic acid sequences that minimizes coamplification of spurious reaction products.